Blood clotting represents part of the protective mechanism in the organism. Induced by a vessel will injury a blood clot is formed to prevent bleeding to death. In addition, vascular diseases, haemostasis and pathological activation of clotting factors may also induce blood clotting. In this case the vessel is obstructed fully or partially, by the intravascular thrombus formed and thrombosis develops. Fibrinolysis represents an other part of the protective mechanism. Here excess blood clots are removed by the enzymes participating in thrombolysis and the dissolution of the thrombus, too.
The blood clotting process is a cascade reaction, a series of catalysed enzyme reactions, where plasma proteins, the so-called clotting factors, are activated consecutively. The factors are marked by Roman numerals, the active form is represented by the letter "a". Trivial names are in use too, thus fibrinogen=factor I (fI), fibrin=factor Ia (fIa), prothrombin=factor II (fII) and thrombin=factor IIa (fIIa). Serine proteases (fXIIa, fVIIa, fXIa, fIXa fXa, fXa and thrombin), some accelerating co-factors (fVa and fVIIIa) and the clottable molecule itself (fibrin) are all formed during the clotting process. fXa and thrombin are the last two factors among the proteases formed. Thrombin, formed upon the action of fXa, initiates the fission of fib-rinogen, resulting in the fibrin clot.
According to the earlier concept of blood clotting mechanism [R. G. MacFarlane, Nature 202, 498 (1964); E. W. Davie and O. D. Ratnoff, Science 145, 1310 (1964)] fX is activated in two ways by an intrinsic and an extrinsic pathway. In the former case the process is initiated by the surface-activated fXII (fXIIa) with the transformation fXI.fwdarw.fXIa which is followed by the reaction fIX.fwdarw.fIXa; fX is activated by fIXa. In the extrinsic pathway the process is initiated by the appearance of the cellular surface receptor, the tissue factor (TF), and the development of the [fVII+TF] or [fVIIa+TF] complex. fX is activated by the [fVIIa+TF] complex.
According to recent findings blood clotting in the living organism is a result of both pathways combined [E. W. Davie et al., Biochemistry 43, 10363 (1991)] where the main steps are the following:
1. In the case of vessel wall injury or disease, TF migrates to the surface and binds a portion of factor VII circulating in the blood. The [fVII+TF]-complex formed is converted by the action of suitable trace amounts of proteases (fXIIa, fXa, fIXa and thrombin) into the active enzyme complex [fVIIa+TF] which activates a small portion of plasma factors IX and X (i.e. small amounts of fIXa and fXa are formed), then it is inactivated by the action of TFPI (Tissue Factor Pathway Inhibitor, earlier name Lipoprotein-Associated Coagulation Inhibitor), the common inhibitor of both fXa and [fVIIa+TF] [T. J. Girard et al., Nature 338, 518-520 (1989)].
2. The fIXa generated together with factor X and cofactor VIIIa produces, in the presence of CA.sup.++ ions, on a phospholipid surface (PL) the "tenase" complex, [fIXa+fVIIIa+fX+PL+CA.sup.++ ], wherein fX is activated to fXa.
3. The fXa generated up to this point, together with prothrombin (fII) and cofactor Va, produces the "prothrombinase complex" [fXa+fVa+fII+PL+CA.sup.++ ], which has a structure similar to that of "tenase". Inside this complex prothrombin is converted to thrombin. The fV.fwdarw.fVa and fVIII.fwdarw.fVIIIa conversions can be performed either by fXa or thrombin.
4. The small amount of thrombin generated converts a portion of fXI to the enzyme fXIa and activates some factors VIII and V, to produce further amounts of fVIIIa and fVa, resp. By now fXIa can carry out the conversion of factor IX to the enzyme fIXa. With this step the chain reaction starting with the Xase-complex and terminated with thrombin formation is resumed. With the repetition of the process increasing amounts of thrombin are formed.
5. At a suitable high thrombin concentration the fibrinogen dissolved in the plasma undergoes partial proteolysis, a fibrin-monomer is generated which is first associated to a soluble fibrin polymer, then it is converted to insoluble fibrin polymer. Here also the thrombin is playing a role, as fXIIla, the factor performing polymerisation, is produced upon its action [L. Lorand and K. Konishi, Arch. Biochem. Biophys. 105, 58 (1964).
The insoluble fibrin polymer is the main component of the blood clot and thrombus, the other being the blood platelet aggregate which is generated primarily upon the action of thrombin, too. The thrombus or blood clot formed entraps the major part of thrombin generated during the process which triggers a new coagulation process when it gets into the solution during the dissolution of the thrombus [A. K. Gash et al., Am. J. Cardiol. 57, 175 (1986); R. Kumar et al., Thromb. Haemost. 72, 713 (1994)].
The above features demonstrate the key role of thrombin in thrombus formation. Consequently all compounds interfering with the function and/or formation of thrombin are of major importance in the therapy of thrombosis
At present the most widely and successfully used compounds applied for the prophylaxis and treatment of thrombosis are the heparins and the vitamin K antagonist coumarins (e.g. Syncumar and Warfarin) which are indirect thrombin inhibitors.
Heparin catalyses the reaction between thrombin and its natural inhibitor, antithrombin-III (AT-III). However, this action of heparin is absent if the plasma concentration of AT-III is lower than 75% of the normal level [R. Egbring et al., Thromb. Haemost. 42, 225 (1979)]. It is also of importance that the thrombin bound by the above-mentioned thrombus fails to be inhibited by this indirect mechanism as it is inaccessible to the heparin-AT-III-complex [J. I. Weitz et al., J. Clin. Invest. 86, 385 (1990)]. In addition, side effects such as treatment-related haemorrhages and thromboembolisms developing due to immunopathological processes are not negligible either [J. M. Walenga et al., Clin. Appl. Thrombosis/Haemostasis, 2(Suppl.1), S21-S27 (1996)].
The vitamin K antagonists may be administered orally, too, their effect is developing after 16-24 hours. They inhibit the development of the reactive forms of some Gla-containing clotting factors (I. e. prothrombin). To achieve therapeutic effects partial inhibition (60-70%) is required [M. P. Esnouf and C. V. Prowse, Biochim. Biophys. Acta 490, 471 (1977)] which can be attained by suitable drug dosage. Vitamin K antagonists, however, are difficult to use due to their narrow therapeutic range, strong dependence on diet composition (vitamin K) and variable individual sensitivity.
The first highly potent synthetic compound directly inhibiting thrombin was the tripeptide aldehyde D-Phe-Pro-Arg-H, a reversible inhibitor, exhibiting significant anticoagulant activity both in vitro and in vivo [S. Bajusz et al., in: Peptides: Chemistry, Structure and Biology (R. Walter and J. Meienhofer, Eds.), Ann Arbor Publ., Ann Arbor, Mich., USA, 603-608 (1975); Int. J. Peptide Protein Res. 12, 217 (1978)]. A series of compounds related to D-Phe-Pro-Arg-H have been synthesised. One of the first was Boc-D-Phe-Pro-Arg-H [S. Bajusz et al., Int. J. Peptide Protein Res. 12, 217 (1978)] and the chloromethylketone analogue (D-Phe-Pro-Arg-CH.sub.2 Cl) which proved to be an irreversible inhibitor [C. Kettner and E. Shaw, Thromb. Res. 14, 969 (1979)]. Further peptides and acylpeptides to be mentioned are the boroarginine analogues (D-Phe- and Boc-D-Phe- as well as Ac-D-Phe-Pro-boroArg) which are potent reversible thrombin inhibitors [C. Kettner et al., J. Biol. Chem. 265, 18289 (1990)] and other analogues of Boc-D-Phe-Pro-Arg-H, including the Boc-D-Chg-Pro-Arg-H analogue [(P. D. Gesellchen and R. T. Shuman, European patent specification No. 0,479,489 A2 (1992)].
In aqueous solutions D-Phe-Pro-Arg-H is prone to undergo spontaneous conversion, but D-MePhe-Pro-Arg-H (GYKI-14766), obtained by methylating the terminal amino group, proved to be of suitable stability while retaining the activity of the parent compound [S. Bajusz et al., U.S. Pat. No. 4,703,036 (1987); J. Med. Chem. 33, 1729 (1990)]. Blood clotting and thrombus formation were significantly inhibited in laboratory animals by the compound [D. Bagdy et al., Thromb. Haemost. 67, 357 and 68, 125 (1992); J. V. Jackson et al., J. Pharm. Exp. Ther. 261, 546 (1992)]; its inhibitory action on the enzymes of fibrinolysis was negligible, co-administered with thrombolytics it significantly promoted the dissolution of the thrombus [C. V. Jackson et al., J. Cardiovascular Pharmacol. 21, 587 (1993)] which could not be attained with the heparin-AT-III complex. Several compounds related to D-MePhe-Pro-Arg-H have been synthesised, e.g. D-MePhg-Pro-Arg-H [R. T. Shuman et al., J. Med. Chem. 36, 314 (1993)].
Anticoagulant activity (i.e. inhibition of the proteolytic reactions in the process) is measured by anticoagulant tests, e.g. in the thrombin time (TT), activated partial thromboplastin time (APTT) and prothrombin time (PT) tests [E. J. W. Bovie et al., Mayo Clinic Laboratory Manual of Haemostasis; W. B. Saunders Co., Philadelphia (1971)]. Plasma, inhibited in spontaneous coagulation, e.g. citrate-plasma, is made to coagulate and the required coagulation time is measured. Upon the action of anticoagulants the coagulation time is prolonged proportionally to the inhibition of the reaction(s) in the process. The anticoagulant effect can be characterised by the substance concentration required to prolong the coagulation time twofold compared to the control (IC.sub.50). The effect of anticoagulants on individual coagulant proteases is measured by the amidolytic method [R. Lottenberg et al., Methods in Enzymol. 80, 341 (1981); G. Cleason, Blood Coagulation and Fibrinolysis 5, 411 (1994)]. The isolated active factor (e.g. thrombin, fXa) and its chromogen or fluorogen peptide-amide substrate are reacted in the presence or absence of the inhibitor, resp. The enzyme inhibiting action is characterised by the inhibitory constant (IC.sub.50) measured during amidolysis.
In the TT test coagulation is initiated by the thrombin added to the citrate plasma. In the system 22 pmol/ml of thrombin is functioning and its inhibition can be measured on the fibrinogen (one of the natural substrates of thrombin) in the presence of plasma components. In the APTT and PT tests the full coagulation process takes place. Depending on the activator fX is activated either by the extrinsic or the intrinsic pathway. The generated fXa activates prothrombin to thrombin which, in turn, triggers plasma coagulation. The coagulation time is prolonged if the enzymes or one of them is inhibited by the inhibitor. In the APTT and PT tests at most 40 pmol/ml Xa can be generated (this is the full amount of factor X present in both systems) while 150 pmol/ml (APTT) and 350 pmol/ml (PT) of thrombin are generated [B. Kaiser et al., Thromb. Res. 65, 157 (1992)].
In the case of D-MePhe-Pro-Arg-H (C1) the concentration prolonging clotting time twofold in the TT, APTT and PT tests amounted to 87, 622 and 2915 nM, resp. These values and the amount of thrombin (22, 150 and 350 pmol/ml) functioning in the tests increased similarly, suggesting that C1 behaves as a thrombin inhibitor in both the APTT and the PT tests and has only slight or no influence on the operation of fXa. In good agreement with these results the amidolytic effect of thrombin on Tos-Gly-Pro-Arg-pNA substrate is inhibited by C1 with an IC.sub.50 =2 nM value, while the amidolytic effect of fXa on the corresponding Bz-Ile-Glu-Gly-Arg-pNA substrate was only slightly affected with an IC.sub.50 =9, 1 mM value (Bajusz et al. Unpublished results).
It is an inherent characteristic of the blood clotting mechanism that the process is inhibited not only by direct thrombin inhibitors but also by factors hindering thrombin formation, e.g. fXa inhibitors. The 60-member polypeptide isolated from tick, TAP (Tick Anticoagulant Peptide) [L. Waksman et al., Science 248, 593 (1990); A. B. Kelly et al., Circulation 86, 1411 (1992)] and DX-9065a (C2), a synthetic non-peptide, (+)-(2S)-2-[4[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-[7[amidino- 2-naphthyl]-propionic acid hydrochloride pentahydrate [T. Hara et al., Thromb. Haemost. 71, 314 (1994); T. Yokoyama et al., Circulation 92, 485 (1995)] are strong inhibitors of blood clotting and thrombus formation. Both the amidolytic effect of fXa and plasma clotting are strongly inhibited in the PT and APTT tests by these compounds, i.e. both the free and the complex-bound Xa factors are equally well inhibited while according to their nature as specific fXa inhibitors thrombin is not inhibited at all, i.e. they fail to exert any activity in the TT test. 4-MeP-Asp-Pro-Arg-H (C3) (international patent application No. 93/15756) and Boc-D-Phe-Nal(1)-Arg-H (C4) (international patent application No. 95/13693) are synthetic peptide inhibitors of fXa.
According to the literature C3 and C4 inhibit the amidolytic activity of fXa on the Z-D-Arg-Gly-Arg-pNA substrate at IC.sub.50 =57 and 30 nM, resp. No data are available on their anticoagulant potency. In our own tests C3 and C4 exhibited also significant inhibition on the Bz-Ile-Glu-Gly-Arg-pNA substrate while their anticoagulant activity proved to be negligible in the plasma clotting tests. The published (C2) and measured (C3-C4) activities of synthetic fXa inhibitors compared to the antithrombin compound C1 are presented in Table 1.
The data of Table 1 demonstrate that in the case of C1 the anticoagulant effect is due to the inhibition of thrombin while in the case of C2 to the inhibition of fXa. The significant fXa inhibitory effect of C3 and C4 is not accompanied by any significant anticoagulant effect. Most probably the fXa active centre in the prothrombinase complex is inaccessible to C3 and C4, these peptides can inhibit only free fXa in solution.
TABLE 1 fXa inhibiting (A) and anticoagulant (B) effect of known synthetic inhibitors A: B: IC.sub.50, .mu.M.sup.a Inhibitor IC.sub.50, nM.sup.b PT APTT TT C1 9133 2.91 0.62 0.09 C2 70 0.52 0.97 NA.sup.c C3 64 19.32 4.59 0.87 C4 86 53.62 9.96 17.24 .sup.a Peptide concentration prolonging clotting time twofold compared to the control in the prothrombin time (PT),activated partial thromboplastin time (APTT) and thrombin time (TT) test .sup.b Value measured with isolated human fXa on Bz-lle-Glu-Gly-Arg-pNA chromogen substrate .sup.c NA = inactive
According to a recent publication [N. A. Prager et al., Circulation 92, 962 (1995)] not only thrombin but also factor Xa, entrapped in the thrombus/blood clot and liberated during dissolution, contributes to the initiation and maintenance of a new coagulation process through the activation of the [fVII+TF]-complex or factors V and VII, resp. Consequently, it is advantageous if the anticoagulants are able to inhibit factor Xa in addition to the inhibition of thrombin, particularly if this inhibition is extended to the clot-bound thrombin and factor Xa, too.